In eukaryotes, gene regulation is largely controlled at the transcriptional level by promoter-specific activator proteins (activators) whose DMA binding sites are typically present upstream of the core promoter of genes transcribed by RNA polymerase II (class II genes). Transcription initiation by RNA polymerase II involves the assembly of general transcription factors (GTFs) on the core promoter to form a preinitiation complex (PIC). A variety of studies indicate that activators work, at least in part, by increasing PIC assembly. Activator mediated stimulation of PIC assembly is believed to result from a direct interaction between the activator and one or more components of the transcription machinery, termed the "target". The unambiguous identification of the direct in vivo targets of activators has been a major challenge in the field. During the past funding period we have developed methods to identify activator targets and study activator-mediated PIC assembly in living cells. In particular, we have shown how chromatin-immunoprecipitation (ChIP) analysis can be used to study in vivo PIC assembly and have developed a robust fluorescence resonance energy transfer (FRET) assay for detecting interactions between transcriptional activation domains (ADs) and their targets in living cells. We will continue to use these methods to identify activator targets and study how activator-target interactions promote PIC assembly. We will also use FRET, in conjunction with other approaches, to delineate the protein interaction network of the PIC and individual multisubunit transcription factors. A central feature of gene regulation in higher eukaryotes is the ability of multiple activators to cooperate with one another to stimulate transcription synergistically. We will use ChIP assays to analyze the mechanistic basis underlying different classes of activator cooperativity. During the past funding period we have identified and characterized a new vertebrate-specific TATA-box-binding protein (TBP)-related factor, TRF3. We will continue to study the role of TRF3 in transcription regulation and normal development. Using diverse experimental systems (mammalian tissue-culture cells, zebrafish, mice), we will identify TRF3 target genes, clone TRF3 interacting proteins, and determine the basis of selective TRF3 function.